Development Of Androgen-independent Prostate Cancer Research Articles

THU496 A Positive Relationship Exists Between CARF And AR In Prostate Cancer

Abstract Disclosure: K.M. Hasan: None. H. Khambati: None. R.R. Huerta: None. J. Vadgama: None. A.P. Sinha-Hikim: None. T.C. Friedman: None. Prostate cancer (PCa) is the second most prevalent cancer among men worldwide. Since PCa onset is androgen dependent, androgen deprivation therapy (ADT) is the first treatment choice for a primary tumor or metastatic PCa. However, ADT eventually leads to the development of castration resistant PCa (CRPC), which is the leading cause of higher mortality in PCa. Thus, there is a need to identify the underlying mechanisms of CRPC to manage this aggressive disease. Increased androgen receptor (AR) expression is one of the most important factors contributing to the development of CRPC. It has been shown that tumor suppressor p53 negatively regulates the expression of AR. So, loss of p53 functions has been associated with increased expression of AR in advanced PCa. However, the mechanism of p53-mediated inhibition of AR is not well understood. In this study, we demonstrated that CARF (CDKN2AIP), a p53 pathway regulatory protein, is highly expressed in PCa, but its significance in PCa development is unknown. Since, like AR, CARF is negatively regulated by p53, we hypothesize that a reciprocal relationship between CARF and p53 could regulate AR expression in PCa cells. Indeed, we found that activation of p53 by DNA damage stress inhibited both CARF and AR in LnCap and 22RV1, AR-positive, and wtp53 cell lines. Our result indicated that CARF and AR were decreased dose-dependent manner, and presumably, that could trigger cell death. Consistent with this speculation, we found that silencing CARF by ShRNA in LnCap triggered apoptosis and inhibition of cell proliferation. With great interest, we further demonstrated that silencing of CARF downregulated the AR level in LnCap and 22RV1 cells suggesting that CARF regulates the expression of AR in PCa. RT-PCR results showed that AR transcripts remained unchanged in CARF-depleted LnCap cells. This result indicated that CARF could regulate the AR stability but not the transcription. Intriguingly, we found that the expression of NEDD4, one of the AR-targeting E3 ligases, was increased in CARF-depleted cells. Therefore, by enhancing the stability, CARF could positively regulate the expression of AR in PCa cells. Further studies are in progress to know more details about the CARF-mediated AR regulation and its impact on androgen-independent PCa development and progression. Presentation: Thursday, June 15, 2023

632P Differential transcriptomic profiling of BCL2-related genes in primary tumor (PT) and metastatic sites (MS) of prostate cancer (PCa)

Bcl-2 anti-apoptotic and pro-apoptotic proteins regulate sensitivity to apoptosis stimuli. Overexpression of Bcl-2 and resistance to apoptosis have been implicated in the development of androgen-independent PCa and disease progression. This project aimed to characterize the transcriptomic profile of BCL2-related genes and investigate distinct sensitivity to apoptosis in PT and MS.

Differential expression of exosomal miRNAs between androgen-dependent prostate cancer cell and androgen-independent prostate cancer cell

Objective Studying the differential expression of exosomal miRNAs between androgen-dependent prostate cancer cell and androgen-independent prostate cancer cell, in order to further elucidate the mechanism of androgen-independent prostate cancer and find new targets for its treatment. Methods Prostate cancer androgen-dependent LNCaP cell and prostate cancer androgen-independent LNCaP-AI+F cell (induced by androgen and flutamide) were selected as the study subjects. Illumina HiSeqTM 2500 was used to perform high-throughput sequencing between the two groups. The differentially expressed exosomal miRNAs was verified by quantitative real-time PCR(qRT-PCR). The difference was statistically significant by t-test. Results Through the analysis of high-throughput sequencing results, thirteen molecules were screened increased in extracellular exosomes of the androgen-independent cell, including miR-7-5p, let-7a-5p, miR-375, miR-423-3p, miR-378a-3p, and miR-92a-3p, etc. Among them, miR-7-5p was verified by quantitative real-time PCR to be up-regulated by 19.52-fold (t=9.857, P=0.001). Conclusion Differentially expressed exosomal miRNAs may predict the development of androgen-independent prostate cancer and may further regulate the development of androgen-independent prostate cancer. Key words: Prostatic neoplasms; Exosomes; microRNAs; High-throughput nucleotide sequencing

Wnt signaling promotes androgen-independent prostate cancer cell proliferation through up-regulation of the hippo pathway effector YAP

Aberrant up-regulation of Wnt/β-catenin signaling is associated with the development and progression of prostate cancer, but the underlying mechanism is unclear. Here we show that in the absence of androgens, the Wnt/β-catenin pathway activates AR-mediated transcription through up-regulation of the Hippo pathway effector Yes-associated protein (YAP). Wnt3a-conditioned medium (Wnt3a-CM) promotes the growth of LNCaP cells and increases AR and YAP protein levels. Moreover, Wnt3a-CM induces the nuclear translocation of YAP and the AR, but not β-catenin, thereby activating the expression of AR- and YAP-dependent genes, in an androgen-independent manner. In addition, depletion of YAP with small interfering RNA (siRNA) prevented Wnt3a-CM-mediated up-regulation of AR-dependent gene expression. Thus, our findings provide mechanistic insight into the proposed cross-talk between the Wnt/β-catenin and Hippo pathways in androgen-independent prostate cancer development.

Inflammation and Nitrosative Stress Effects in Ovarian and Prostate Pathology and Carcinogenesis.

Prostate and ovarian cancers are major contributors to cancer-related deaths worldwide. Recently, inflammation and nitrosative stress have been implicated in carcinogenesis, with the overexpression of NOS2 and concomitant release of nitric oxide (NO) associated with cancer initiation and progression. Recent Advances: An increasing body of evidence indicates an association between NOS2 expression and aggressive ovarian cancer. Research also indicates a role for NO in prostate disease pathology and prostate cancer. A therapeutic role for NOS2 inhibition and/or NO drugs exists for the treatment of both ovarian and prostate tumors. Herein, we review the key molecular effects associated with NOS2 in ovarian and prostate cancer. NOS2 increases angiogenesis and tumor proliferation and correlates with aggressive type II ovarian tumors. NOS2 expressing tumors are sensitive to cisplatin chemotherapy, and NO may be used to sensitize cisplatin-resistant tumors to chemotherapy. NOS2 is highly expressed in prostate tumors compared to non-neoplastic prostate pathologies. NO may play a role in the development of androgen-independent prostate cancer via s-nitrosylation of the androgen receptor. Moreover, NOS2 inhibitors and NO donor drugs show therapeutic potential in ovarian and prostate cancer as single agents or dual drugs, by either inhibiting the effects of NOS2 or increasing NO levels to induce cytotoxic effects. NOS2 and NO present new targets for the treatment of ovarian and prostate tumors. Furthermore, understanding NO-related tumor biology in these cancers presents a new means for improved patient stratification to the appropriate treatment regimen. Antioxid. Redox Signal. 26, 1078-1090.

Clinicopathological and Prognostic Factors in 106 Prostate Cancer Patients Aged ≤55 Years: A Single-Center Study in China.

BackgroundEarly-onset prostate cancer patients (aged ≤55 years) from Western countries have been well characterized in previous studies. However, the clinicopathological and prognostic characteristics of early-onset Chinese prostate cancer patients have not yet been assessed. This study aimed to examine the clinicopathological and prognostic factors of prostate cancer patients aged ≤55 years in a single Chinese center.Material/MethodsOne hundred six prostate cancer patients aged ≤55 years with complete clinicopathological data who were treated at our hospital between January 2000 and June 2014 were selected for this study. Survival rate was investigated by Kaplan-Meier analysis, and prognostic factors were examined by univariate and multivariate analysis.ResultsThe median time from the onset of symptoms to diagnosis was 3.5 months (range, 2–55 months). The median time after endocrine therapy to development of androgen-independent prostate cancer was 10.5 months. A total of 54 patients died (50.9%), of whom 96.2% died from prostate cancer. The 1-, 3-, and 5-year overall survival rates were 88.7%, 66.2%, and 36.0%, respectively. Univariate and multivariate analysis showed that T staging, visceral metastasis, pathological pattern, and Gleason sum were independent prognostic factors in these patients.ConclusionsProstate cancer patients aged ≤55 years are often omitted or misdiagnosed in China. Furthermore, the pathology patterns in this age group were mostly complicated with a high degree of malignancy. Late staging, visceral metastasis, pathological pattern, and high Gleason score were independent prognostic factors in these patients. Comprehensive therapy combined with local therapy is an effective treatment strategy.

Abstract A16: Different epigenetic mechanisms involved in the regulation of SFRP1 gene in prostate cancer

Abstract Among men, prostate cancer (PCa)[1] is the second most common cancer and the fifth cause of death worldwide [2]. Androgens play an important role in the development of the disease. For advanced PCa the standard therapy is androgen depletion. However, after 2 or 3 years a high percentage of patients become resistant to this therapy and castration resistant prostate cancer (CRPC) is developed. There is no successful treatment for CRPC, leading into death [3, 4]. Wingless pathway (WNT) is aberrantly activated in several cancer types and in PCa it is involved in AR activity modulation, promoting cancer development [5]. Secreted Frizzled Related Protein 1 (SFRP1) is commonly known as a WNT inhibitor and generally proposed as a tumor suppressor gene. SFRP1 can exert its tumor suppressor activity by inhibiting the AR transcriptional activity [6]. Usually in cancer, SFRP1 mRNA is down regulated and this is associated to increased DNA methylation at its promoter region [7]. In contrast, in PCa there are different reports suggesting SFRP1 repression in absence of DNA methylation [8]. In this work we aimed to investigate two different epigenetic mechanisms for SFRP1 silencing in PCa. For the in vitro model, we used normal prostate cells RWPE-1 and PCa cell lines: LNCaP, 22RV1 and PC3. We confirmed the correlation between mRNA expression loss and the increased DNA methylation of SFRP1 only in PC3 cells, but not in LNCaP and 22RV1, were instead of DNA methylation we found enrichment of H3K27me3, the Polycomb Repressive Complex 2 (PRC2) mark. To correlate this findings, we analyzed the expression of SFRP1 in fresh frozen tissue samples, 11 primary PCa, 10 Benign Prostatic Hyperplasia (BPH) and 4 normal prostates (NP). We found SFRP1 downregulation in all PCa and BPH samples compared to Normal Prostate, but DNA methylation was increased only in PCa but not in BPH, this suggests a novel repressive mechanism for SFRP1 independent of DNA methylation. To see if AR has a role in regulating mRNA SFRP1 expression, we silenced AR with a siRNA, but no SFRP1 expression levels were modified, so we concluded AR was not directly involved in SFRP1 regulation. All these data together suggest different repression mechanisms for SFRP1, one depending on DNA methylation and another one depending on H3K27me3 PRC2 repressive mechanism, Androgen receptor is not directly involved in SFRP1 downregulation, so other factors might be involved in SFRP1 expression loss in PCa.

Abstract B1-40: Biological network analysis using an in vitro model of androgen-resistance in prostate cancer

Abstract The development of androgen resistance is a major limitation to androgen deprivation treatment in prostate cancer (PCa). We have developed an in vitro model of androgen-resistance using the androgen sensitive cell line LNCaP to characterize the phenotypic and transcriptomic changes occurring as androgen resistance develops. Our aim is to understand biological network profiles of transcriptomic changes during the transition to androgen-resistance and to validate these changes between our in vitro model and previously published clinical datasets (paired samples before and after androgen-deprivation therapy of patients with advanced PCa) (1). Methods: PCa cells, LNCaP, expressing mutated AR which are androgen-dependent (2,3), are used in the development of an androgen-resistant subline. Subline cells were established by prolonged cultures in media + 10% CS-FBS to mimic the clinical course of PCa. Cell proliferation, cell motility and invasion, morphology, AR expression were examined. RNA-sequencing was performed using the parental LNCaP cells and an androgen-resistant subline (LNCaP-AI) established by chronic exposure to the androgen-deprivation. Reads from cells and clinical samples (1), pre- vs. post-treatment, were processed through the same standard pipeline and quality control. Data outputs were analysed as differential expression [DEG] (EdgeR) (4) and top scoring protein-protein interaction (PPI) networks [PINA2 (5) and BioNet (6)]. Data outputs from the cell line model and clinical samples were compared. Results: LNCaP cells initially showed poor growth after prolonged exposure to androgen-deprived conditions but later adapted and started to grow well. After 24 weeks of androgen-deprivation, LNCaP-AI's growth was no longer responsive to addition of androgen [0.1 - 10 nM]. AR expression was not different in LNCaP and LNCaP-AI (P>0.05). LNCaP-AI cells had increased proliferation and cell invasion compared to LNCaP. We identified key genes that overlap between our cell line and clinical RNAseq (1) datasets and analyzed the overlapping PPI network that showed the same pattern of behavior in both datasets. The network revealed several potential mechanisms and gene interactions that warrant further investigation, including cooperative behaviors of other nuclear receptors, TP63 mediated signalling pathway and Aryl hydrocarbon receptor transcriptional pathway. Conclusion: Cell line model of androgen-resistance will be used for further longitudinal study of the mechanism of castrate resistant prostate cancer (CRPC). Our approach allows for better characterization of biological processes of CRPC. Knowledge of the genetic profiles during transition to androgen resistance will improve our understanding of this common clinical scenario and may lead to biomarker discovery.

Prostate cancer: considering the treatment options

Nursing and Residential CareVol. 16, No. 10 EditorialProstate cancer: considering the treatment optionsIan PeateIan PeateSearch for more papers by this authorIan PeatePublished Online:17 Sep 2014https://doi.org/10.12968/nrec.2014.16.10.548AboutSectionsView articleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareShare onFacebookTwitterLinked InEmail View article References Cancer Research UK (2014a) Prostate cancer incidence statistics. http://tinyurl.com/kxozyqh (accessed 21 August 2014) Google ScholarCancer Research UK (2014b) Prostate cancer statistics. http://tinyurl.com/ovphmtx (accessed 21 August 2014) Google ScholarCancer Research UK (2014c) Enzalutamide (Xtandi). http://tinyurl.com/lk2cxsn (accessed 21 August 2014) Google ScholarElectronic Medicines Compendium (2014) Enzalutamide. http://tinyurl.com/kl66xxl (accessed 21 August 2014) Google ScholarFeldman BJ, Feldman D (2001) The development of androgen-independent prostate cancer. Nat Rev Cancer 1(1): 34–45 Crossref, Google ScholarNational Institute for Health and Care Excellence (2014) NICE: Diagnosing and Treating Prostate Cancer. http://www.nice.org.uk/Guidance/CG175 (accessed 21 August 2014) Google ScholarNursing and Midwifery Council (2008) The Code: standards of conduct, performance and ethics for nurses and midwives. http://tinyurl.com/35om3cs (accessed 21 August 2014) Google ScholarProstate Cancer UK (2014) NICE's decision on enzalutamide means that some men will still miss out. http://tinyurl.com/kfn4pd6 (accessed 21 August 2014) Google ScholarScher HI, Fizazi K, Saad F et al. (2012) Increased survival with enzalutamide in prostate cancer after chemotherapy. N Engl J Med 367(13): 1187–97 Crossref, Google Scholar FiguresReferencesRelatedDetails 2 October 2014Volume 16Issue 10ISSN (print): 1465-9301ISSN (online): 2052-2932 Metrics History Published online 17 September 2014 Published in print 2 October 2014 Information© MA Healthcare LimitedPDF download

Targeting the relaxin hormonal pathway in prostate cancer.

Targeting the androgen signalling pathway has long been the hallmark of anti-hormonal therapy for prostate cancer. However, development of androgen-independent prostate cancer is an inevitable outcome to therapies targeting this pathway, in part, owing to the shift from cancer dependence on androgen signalling for growth in favor of augmentation of other cellular pathways that provide proliferation-, survival- and angiogenesis-promoting signals. This review focuses on the role of the hormone relaxin in the development and progression of prostate cancer, prior to and after the onset of androgen independence, as well as its role in cancers of other reproductive tissues. As the body of literature expands, examining relaxin expression in cancerous tissues and its role in a growing number of in vitro and in vivo cancer models, our understanding of the important involvement of this hormone in cancer biology is becoming clearer. Specifically, the pleiotropic functions of relaxin affecting cell growth, angiogenesis, blood flow, cell migration and extracellular matrix remodeling are examined in the context of cancer progression. The interactions and intercepts of the intracellular signalling pathways of relaxin with the androgen pathway are explored in the context of progression of castration-resistant and androgen-independent prostate cancers. We provide an overview of current anti-hormonal therapeutic treatment options for prostate cancer and delve into therapeutic approaches and development of agents aimed at specifically antagonizing relaxin signalling to curb tumor growth. We also discuss the rationale and challenges utilizing such agents as novel anti-hormonals in the clinic, and their potential to supplement current therapeutic modalities.

Abstract 2126: Inhibition of hedgehog and androgen receptor signaling pathways produced synergistic suppression of androgen independent prostate cancer progression.

Abstract Introduction: Prostate cancer (PCa) is the second most common cause of death from cancer and the leading cancer diagnosed in the US men. Approximately 1 in 6 American men will be diagnosed with prostate cancer during his lifetime. Estimated new cases and deaths from prostate cancer in the United States for 2012 include: 241,740 new cases and 28,710 deaths. Initial treatment for metastatic PCa involves androgen ablation therapy, which causes regression of androgen-dependent tumors. However they all eventually relapse resulting in recurrent androgen independent prostate cancer (AIPC). Methods: This study aims at assessing the role of Hedgehog (Hh) and Androgen receptor (AR) signaling in the development of AIPC. Quantitative RT-PCR analysis was performed for AIPC cells in vitro and for AIPC xenograft after castration, to assess the levels of Hh and AR signaling components. Hh inhibitors, LDE225 (Novartis) and Cyclopamine, and Androgen receptor inhibitor Pyrvinium pamoate were tested individually and in combination to assess the suppression of AIPC progression both in vitro and in vivo. AR-responsive and Gli-responsive promoter-luciferase assay and MTT assay were used for in vitro studies. Intraperitoneal administration of drugs and bioluminescence imaging for monitoring orthotopic tumors in mice were used to study the effect of drugs on the growth of AIPC in vivo. Results: We demonstrate that androgen deprivation leads to increase in Hedgehog (Hh) signaling components in the AIPC cells in vitro and in vivo. AIPC cells also show increased sensitivity to low concentrations of androgen, suggesting increased androgen receptor expression. Together, these results suggest that increased Hh signaling and androgen receptor expression in the absence of androgen agonist plays an important role in the development and progression of AIPC. Our results show that inhibiting either Hh or androgen receptor signaling pathway individually was not effective in preventing AIPC cell proliferation in vitro as well as in inhibiting xenograft tumor growth in mice. In contrast, combination of an Hh pathway inhibitor with an androgen receptor inhibitor synergistically suppressed the growth of AIPC cells in vitro as assessed by MTT assay and reduced tumor burden in a mouse xenograft model of AIPC as compared to individual treatments.Conclusion: Upregulated Hh signaling and AR expression mediates development and progression of AIPC. Combined inhibition of Hh and androgen receptor signaling pathways may be a novel effective therapeutic strategy for the treatment of androgen independent prostate cancer. Citation Format: pramod gowda, Jianhong Deng, Sweta Mishra, LuZhe Sun. Inhibition of hedgehog and androgen receptor signaling pathways produced synergistic suppression of androgen independent prostate cancer progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2126. doi:10.1158/1538-7445.AM2013-2126

Screening of membrane antigen differentially expressed in androgen-dependent prostate cancer and androgen-independent prostate cancer

To determine the screening of the expression of membrane proteins in androgen-dependent prostate cancer (ADPC) and androgen-independent prostate cancer (AIPC) and to explore the mechanism of membrane proteins in these two cancers. Serum samples were collected from 3 patients with ADPC and another 3 patients with AIPC. The serum was incubated with ADPC cell line LNCaP and/or AIPC cell line PC-3 and detected by immunoprecipitation and Western blot. Differentially expressed proteins between ADPC and AIPC identified by mass spectrometry were compared and their expression level and location were analyzed by immunofluorescence. Altogether 11 membrane proteins were identifited, such as the Neural-Cadherin precursor, ER60 precursor, Claudin-4, and so on. Immunofluorescence revealed that the expression level of Claudin-4 in PC-3 cells was higher than in LNCaP cells. We can use the screening method to study membrane proteins in prostate cancer. Claudin-4 may play an important role in the pathogenesis and the development of AIPC.

Molecular Alterations during Progression of Prostate Cancer to Androgen Independence

Prostate cancer is the most commonly diagnosed cancer among men in North America and is a leading cause of death. Standard treatments include androgen deprivation therapy, which leads to improved clinical outcomes. However, over time, most tumors become androgen independent and no longer respond to hormonal therapies. Several mechanisms have been implicated in the progression of prostate cancer to androgen independence. Most tumors that have become androgen independent still rely on androgen receptor (AR) signaling. Mechanisms that enhance AR signaling in androgen-depleted conditions include: AR gene amplification, AR mutations, changes in the balance of AR cofactors, increases in steroidogenic precursors, and activation via "outlaw" pathways. Along with AR signaling, various other AR-independent "bypass" pathways have been shown to operate aberrantly during androgen independence. Changes in the epigenetic signatures and microRNA concentrations have also been implicated in the development of androgen-independent prostate cancer. Understanding of the molecular mechanisms that lead to the development of androgen-independent prostate cancer will allow for improved therapeutic strategies that target key pathways and molecules that are essential for these cells to survive.

Androgen Depletion Induces Senescence in Prostate Cancer Cells through Down-regulation of Skp2

Although the induction of senescence in cancer cells is a potent mechanism of tumor suppression, senescent cells remain metabolically active and may secrete a broad spectrum of factors that promote tumorigenicity in neighboring malignant cells. Here we show that androgen deprivation therapy (ADT), a widely used treatment for advanced prostate cancer, induces a senescence-associated secretory phenotype in prostate cancer epithelial cells, indicated by increases in senescence-associated β-galactosidase activity, heterochromatin protein 1β foci, and expression of cathepsin B and insulin-like growth factor binding protein 3. Interestingly, ADT also induced high levels of vimentin expression in prostate cancer cell lines in vitro and in human prostate tumors in vivo. The induction of the senescence-associated secretory phenotype by androgen depletion was mediated, at least in part, by down-regulation of S-phase kinase-associated protein 2, whereas the neuroendocrine differentiation of prostate cancer cells was under separate control. These data demonstrate a previously unrecognized link between inhibition of androgen receptor signaling, down-regulation of S-phase kinase-associated protein 2, and the appearance of secretory, tumor-promoting senescent cells in prostate tumors. We propose that ADT may contribute to the development of androgen-independent prostate cancer through modulation of the tissue microenvironment by senescent cells.

Abstract LB-381: Inhibition of the hedgehog signaling pathway has significant preclinical activity and prevents development of androgen-independent prostate cancer

Abstract Introduction: Prostate cancer (PCa) is the second leading cause of cancer death in American men. Patients with advance PCa are often treated first with anti-androgen therapy, but will invariably develop androgen-independent prostate cancer (AIPC). A recent study has shown that androgen deprivation can lead to increased expression of hedgehog signaling components. Hedgehog signaling is also known for normal and cancer stem cell self-renewal. Therefore, in this study, we tested the hypothesis that the inhibition of hedgehog pathway with a novel hedgehog inhibitor, LDE225 (Novartis®), will block the development and growth of AIPC. Methods and Results: Two androgen-dependent PCa (ADPC) cell lines were adapted to grow in an androgen-free culture medium. The emergence of AIPC cells was found to be associated with increased expression of hedgehog ligands, and hedgehog signaling activity through the activation of its downstream Gli transcription factors as detected using a Gli-responsive promoter luciferase reporter. We also found that treatment with LDE225 was more effective in inhibiting the growth of AIPC cells than ADPC cells. Furthermore, the treatment with LDE225 also inhibited the emergence of AIPC cells when the ADPC cells were cultured in the androgen-free medium. Finally, treatment of orthotopic xenograft PCa model with LDE225 is underway to confirm our pre-clinical in-vitro activity in both ADPC and AIPC models. Conclusion: These results support our hypothesis that the activation of hedgehog pathway contributes to the development of AIPC and provide the rationale and guidance for design of clinical trials utilizing LDE225 in PCa patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-381. doi:10.1158/1538-7445.AM2011-LB-381

MicroRNA-616 Induces Androgen-Independent Growth of Prostate Cancer Cells by Suppressing Expression of Tissue Factor Pathway Inhibitor TFPI-2

Expression of microRNA genes is profoundly altered in cancer but their role in the development of androgen-independent prostate cancer has received limited attention as yet. In this study, we report a functional impact in prostate cancer cells for overexpression of the microRNA miR-616, which occurred consistently in cells that were androgen-independent (AI) versus androgen-dependent (AD). miR-616 overexpression was confirmed in malignant prostate tissues as opposed to benign prostate specimens. Stable miR-616 overexpression in LNCaP cells by a lentiviral-based approach stimulated AI prostate cancer cell proliferation in vitro whereas concomitantly reducing androgen-induced cell growth. More importantly, miR-616 overexpressing LNCaP cells overcame castration resistance as shown by an enhanced ability to proliferate in vivo after bilateral orchiectomy. Conversely, antagonizing miR-616 in AI prostate cancer cells yielded opposite effects. Microarray profiling and bioinformatics analysis identified the tissue factor pathway inhibitor TFPI-2 mRNA as a candidate downstream target of miR-616. In support of this candidacy, we documented interactions between miR-616 and the 3'UTR of TFPI-2 and determined TFPI-2 expression to be inversely correlated to miR-616 in a series of prostate cell lines and clinical specimens. Notably, reexpression of TFPI-2 in LNCaP cells with stable miR-616 overexpression rescued the AD phenotype, as shown by a restoration of androgen dependence and cell growth inhibition. Taken together, our findings define a functional involvement for miR-616 and TFPI-2 in the development and maintenance of androgen-independent prostate cancer.

Characterization of the small RNA transcriptomes of androgen dependent and independent prostate cancer cell line by deep sequencing.

Given the important roles of miRNA in post-transcriptional regulation and its implications for cancer, characterization of miRNA facilitates us to uncover molecular mechanisms underlying the progression of androgen-independent prostate cancer (PCa). The emergence of next-generation sequencing technologies has dramatically changed the speed of all aspects of sequencing in a rapid and cost-effective fashion, which can permit an unbiased, quantitive and in-depth investigation of small RNA transcriptome. In this study, we used high-throughput Illumina sequencing to comprehensively represent the full complement of individual small RNA and to characterize miRNA expression profiles in both the androgen dependent and independent Pca cell line. At least 83 miRNAs are significantly differentially expressed, of which 41 are up-regulated and 42 are down-regulated, indicating these miRNAs may be involved in the transition of LNCaP to an androgen-independent phenotype. In addition, we have identified 43 novel miRNAs from the androgen dependent and independent PCa library and 3 of them are specific to the androgen-independent PCa. Function annotation of target genes indicated that most of these differentially expressed miRNAs tend to target genes involved in signal transduction and cell communication, epically the MAPK signaling pathway. The small RNA transcriptomes obtained in this study provide considerable insights into a better understanding of the expression and function of small RNAs in the development of androgen-independent prostate cancer.

Estrogen receptor β and the progression of prostate cancer: role of 5α-androstane-3β,17β-diol

Prostate cancer (PC) develops in response to an abnormal activation of androgen receptor induced by circulating androgens and, in its initial stages, is pharmacologically controlled by androgen blockade. However, androgen ablation therapy often allows androgen-independent PC development, generally characterized by increased invasiveness. We previously reported that 5alpha-androstane-3beta,17beta-diol (3beta-Adiol) inhibits the migration of PC cell lines via the estrogen receptor beta (ERbeta) activation. Here, by combining in vitro assays and in vivo imaging approaches, we analyzed the effects of 3beta-Adiol on PC proliferation, migration, invasiveness, and metastasis in cultured cells and in xenografts using luciferase-labeled PC3 (PC3-Luc) cells. We found that 3beta-Adiol not only inhibits PC3-Luc cell migratory properties, but also induces a broader anti-tumor phenotype by decreasing the proliferation rate, increasing cell adhesion, and reducing invasive capabilities in vitro. All these 3beta-Adiol activities are mediated by ERbeta and cannot be reproduced by the physiological estrogen, 17beta-estradiol, suggesting the existence of different pathways activated by the two ERbeta ligands in PC3-Luc cells. In vivo, continuous administration of 3beta-Adiol reduces growth of established tumors and counteracts metastasis formation when PC3-Luc cells are engrafted s.c. in nude mice or are orthotopically injected into the prostate. Since 3beta-Adiol has no androgenic activity, and cannot be converted to androgenic compounds, the effects here described entail a novel potential application of this agent against human PC.

1289 A MECHANISM FOR ANDROGEN-INDEPENDENT PROSTATE CANCER THROUGH MODULATION OF ANDROGEN RECEPTOR SIGNALING BY THE P21-ACTIVATED KINASE 6 (PAK6)

You have accessJournal of UrologyProstate Cancer: Basic Research VII1 Apr 20101289 A MECHANISM FOR ANDROGEN-INDEPENDENT PROSTATE CANCER THROUGH MODULATION OF ANDROGEN RECEPTOR SIGNALING BY THE P21-ACTIVATED KINASE 6 (PAK6) Min Zhang, Michael Siedow, Gregory Saia, and Arnab Chakravarti Min ZhangMin Zhang More articles by this author , Michael SiedowMichael Siedow More articles by this author , Gregory SaiaGregory Saia More articles by this author , and Arnab ChakravartiArnab Chakravarti More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.873AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES p21-activated kinase 6 (PAK6) is a serine/threonine kinase belonging to the p21-activated kinase (PAK) family. PAK6 was first identified in a yeast two-hybrid screen for novel androgen receptor (AR) interactors, but its functions have not been established. In this study, we explored the role of PAK6 in mediation hormone resistance in prostate cancer cells. METHODS PAK6 expression was detected in androgen deprived prostate cancer cell line LNCaP. Short hairpin RNA (shRNA) was used to attenuate PAK6 expression in LNCaP cells, and apoptosis and cell cycle distribution were assessed in these cells. Given that AR can be phosphorylated by either the AKT or the mitogen-activated protein kinase (MAPK) pathway and becomes a ligand-independent AR, we investigated the effect of PAK6 on the cross-talks between AR signaling and AKT/MAPK pathways. RESULTS We found that PAK6 expression in LNCaP PCa cells was markedly increased when the cells were cultured for 8 weeks in steroid hormone depleted medium. Antagonism of PAK6 using short hairpin RNA (shRNA) significantly increased antiangogen bicalutamide-induced apoptosis and G1 arrest. These finds implied that PAK6 may play a role in the development of androgen independent prostate cancer. Knockdown of PAK6 in LNCaP cells diminished AKT phosporylation at Ser473 and Thr308 as well as p44/42 MAPK phophorylation at Thr202 and Tyr204, indicating that overexpression of PAK6 during androgen deprivation may activate AR signaling by phosporylation of both AKT and MAPK. CONCLUSIONS These findings extended the current vision of PAK6 functions, and indicated that PAK6 may be involved in androgen-independent prostate cancer development through modulation of AR signaling. Columbus, OH© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e499 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Min Zhang More articles by this author Michael Siedow More articles by this author Gregory Saia More articles by this author Arnab Chakravarti More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Comparison of Gene Expression in LNCaP Prostate Cancer Cells after Treatment with Bicalutamide or 5-Alpha-Reductase Inhibitors

Introduction: Androgen deprivation is the preferred treatment for disseminated prostate cancer. However, it mostly leads to the development of incurable androgen-independent disease. The aim of the present study was to compare gene expression changes that occur after treatment with either the antiandrogen bicalutamide or the 5-α-reductase inhibitors finasteride (MK906) and MK386. Materials and Methods: LNCaP cells of low passages were treated with MK906 and MK386 at 5 µM each or with bicalutamide at 10 µM for 48 h. In these cultures we analyzed the expression of 22,500 transcripts on the Affymetrix Human U133+ 2.0 GeneChip platform. Gene expression was verified by real-time quantitative Taqman PCR. Results: Our studies revealed 312 differentially regulated genes upon bicalutamide treatment and 68 differentially regulated genes upon treatment with the 5-α-reductase inhibitors. There were 35 genes equally regulated by both drugs. This subset of genes included those with the highest fold change in both treatment groups. In the subset KlK2, TMPRSS2, TRGC2, PMEPA1 and TM4SF1 were downregulated, whereas EGR1, DDC and OPRK1 were upregulated. Conclusions: A cohort of interesting genes that are differentially expressed after androgen withdrawal could be found in this study. Investigation into these genes could contribute to a better understanding of antiandrogen treatment and development of androgen-independent prostate cancer.